Summary Tissue homeostasis and regeneration are sustained by stem cells, which must both continually renew themselves while keeping up with the demand of new differentiated cell types to replace those lost throughout the life of a tissue. Failure to properly balance these two processes can lead to a host of problems, including impaired organ function and tissue repair, as well as tissue overgrowth and cancer. Understanding the basic mechanisms underlying this delicate equilibrium of stem cell self-renewal and differentiation is an essential step in preventing and treating a wide range of diseases. We still lack fundamental knowledge of how these stem cell behaviors are regulated during tissue regeneration in an intact, living tissue. The goal of this proposal is to understand how individual stem cell decisions are made within the stem cell population so that collectively their behaviors remain balanced within the broader context of the tissue, while preserving both structure and function. The challenge in addressing these fundamental questions lies in the inability to study these dynamic cellular processes in an intact mammal. To this end, my laboratory has established an in vivo strategy to directly visualize and manipulate stem cells in the skin epithelium of live mice, taking advantage of its unique accessibility, continuous regeneration throughout its lifetime, and the presence of multiple stem cell populations that use distinct regenerative strategies. Using a novel, non-invasive two-photon imaging approach, we are finally able to follow stem cells as they self-renew and differentiate over time. In order to understand how stem cell behaviors are balanced , and determine how a stem cell's shape, position and molecular character affect cell fate decisions and the overall regenerative ability of the tissue, we have combined our imaging approach with newly developed methods that allow us to manipulate each cell and their behavior individually in a live mouse. This unique combination of expertise, knowledge, and development of novel in vivo imaging tools has allowed us to first, define aspects of tissue regeneration that were previously inaccessible, revealing unexpected principles of stem cells and second, to understand the complex interplay between stem cell behaviors that fuel tissue regeneration over time. The goal of this proposal is to advance our understanding of how stem cell behaviors are balanced to sustain tissue regeneration over time through the use of an integrated approach of cutting edge imaging technology, genetic manipulation and cell biology. Given that many aspects of stem cell biology have been shown to be widely conserved in other organs, our findings will be relevant to other tissues as well, and will provide an important foundation for the treatment of a range of diseases.